The objective of the proposed research is to understand how the structure and function of membrane-spanning channels is determined by their amino acid sequence and by the host bilayer. This question will be examined using the linear gramicidins- a model system that continues to offer a combination of advantages that continues to set it apart from other ion channels: the channel structure is known at atomic resolution, which guides the experimental design and data analysis; one can obtain structural information using single-channel measurements; the channels are permeable only to monovalent cautions, such that single-channel measurements provide direct information about the channels catalytic ability; the channel-forming molecules are made/modified using peptide chemical methods, which allows for convenient introduction of non-genetic amino acids; and gramicidin channels are suitable as molecular force transducers to quantify the energetics of channel/membrane interactions, which provides for a novel way to examine membrane/protein interactions. The experiments address the following questions: what are the molecular determinants of channel folding, membrane insertion, and stability; how does a structural destabilization introduce voltage-dependent gating; how is channel structure and function modulated by the host bilayer; and what are the molecular and structural determinants of the channels' permeability characteristics? These questions will be examined using a combination of single- channel experiments and molecular modeling: single-channel current transitions characterize the channels' catalytic efficiency; the formation of heterodimeric channels, and their relative stability and appearance rates, constitutes a new method to elucidate structural questions pertaining to channel folding, which is especially useful for detecting rare conformers. The experimental results will guide conformational energy and molecular dynamics calculations, which in turn will be used to interpret the experimental results and guide the design of new experiments. The aims are to understand how the amino acid sequence and the membrane environment interact in determining channel structure and function; how a stress introduced by sequence modifications affect the structure and dynamics; how alterations in the host bilayer alter channel function; and how the rate of ion movement, and the ion selectivity, is determined by the channel structure and amino acid sequence.